The cytokine IFN-gamma is essential for host defense against intracellular pathogens and the development of IFN-gamma producing Th1 lymphocytes plays a critical role in control of these infectious agents. If left unregulated, however, the Th1 immune response can cause serious damage to host tissues and lead to mortality. In the case of Toxoplsma gondii infection, we have previously demonstrated that the induction of mediators that can regulate IFN-gamma effector functions are as important for host survival as the expression of IFN-gamma itself. Thus, mice deficient in IL-10 succumb during the acute phase of infection, despite enhanced parasite control. Subsequently we have demonstrated that this host-protective IL-10 derives in autocrine fashion from conventional IFN-gamma producing T-bet+ Foxp3neg Th1 cells, which simultaneously display both effector and regulatory functions. Our current research in this area focuses on the molecular mechanisms that regulate IL-10 expression in Th1 cells. We have confirmed that expression of IL-10 in Th1 lymphocytes from T. gondii-infected mice is regulated at the level of transcription, and by microarray compared the expression of other genes in IL-10- and IL-10+ Th1 cells as well as the status of chromatin modification in the IL-10 gene locus. Several transcription factors (e.g. TCF-1) and signaling pathways (e.g. STAT) have so far been identified as associated with the differential expression of IL-10. At present we are testing their relevance by employing either knock-out mouse strains for the corresponding genes or by using bone-marrow chimeras. Our results indicate that, while development of IL-10+ Th1 cells is both IL-21- and Stat-3-independent, it is partially dependent on intact signaling through IL-27R or Stat-1 in CD4+ T lymphocytes. We are also comparing the life span of IL-10+ and IL-10neg Th1 cells in order to determine if IL-10 production is a property of terminally differentiated Th1 cells, and whether IL-10+ Th1 cells can establish long-term memory. IL-10+IFN-gamma+ CD4 T cells can be readily detected in the blood of healthy individuals and thus, as an extension of the murine studies described above, we have started to analyze this Th population in human blood samples. In particular, since IL-10 is known to contribute to suppression of HIV immune response, we will analyze blood specimens from HIV+, T. gondii and co-infected individuals for the presence of IL-10+ Th1 cells. We will also test whether their frequency is altered following anti-retroviral therapy. During the report period we have made the new observation that glucocorticoid receptor (GR) expression in T lymphocytes also plays a critical regulatory role in preventing immunopathology during T. gondii infection. Induction of glucorticoids is responsible for the selective loss of double CD4+ CD8+ thymocytes and contributes to the dramatic thymic atrophy ( 90%) observed on day 8 after i.p. injection of either type I or type II strains of the parasite. However, neither soluble extract nor irradiated parasites trigger involution of thymus, indicating a requirement for active infection. The GR knock-out animals which are deficient in GR expression in CD4+ and CD8+ T cells display acute mortality after T. gondii infection and this is associated with an exacerbated systemic IFN-gamma;response, despite no overt changes in parasite load or the responsiveness of innate immune cells as evaluated by serum IL-12 and IL-27 production. Our preliminary results indicate that enhanced IFN-gamma responsiveness by both CD4 and CD8 contributes to the decreased survival of the infected mice. The glucocorticoid-dependent negative feed-back loop may selectively target CD8 T lymphocytes since depletion of CD4 T cells that abrogates systemic IL-10 production and has no significant effect on the survival of infected WT mice, fails to rescue the acute mortality of infected GR KO animals and results in even higher serum IFN-gamma levels than in control or anti-CD8 mAb treated T. gondii-infected GR knock-out mice.